Abstract
Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomatids. Its major components are several thousand topologically interlocked DNA minicircles. Their replication origins are recognized by universal minicircle sequence-binding protein (UMSBP), a CCHC-type zinc finger protein, which has been implicated with minicircle replication initiation and kDNA segregation. Interactions of UMSBP with origin sequences in vitro have been found to be affected by the protein's redox state. Reduction of UMSBP activates its binding to the origin, whereas UMSBP oxidation impairs this activity. The role of redox in the regulation of UMSBP in vivo was studied here in synchronized cell cultures, monitoring both UMSBP origin binding activity and its redox state, throughout the trypanosomatid cell cycle. These studies indicated that UMSBP activity is regulated in vivo through the cell cycle dependent control of the protein's redox state. The hypothesis that UMSBP's redox state is controlled by an enzymatic mechanism, which mediates its direct reduction and oxidation, was challenged in a multienzyme reaction, reconstituted with pure enzymes of the trypanosomal major redox-regulating pathway. Coupling in vitro of this reaction with a UMSBP origin-binding reaction revealed the regulation of UMSBP activity through the opposing effects of tryparedoxin and tryparedoxin peroxidase. In the course of this reaction, tryparedoxin peroxidase directly oxidizes UMSBP, revealing a novel regulatory mechanism for the activation of an origin-binding protein, based on enzyme-mediated reversible modulation of the protein's redox state. This mode of regulation may represent a regulatory mechanism, functioning as an enzyme-mediated, redox-based biological switch.
Highlights
A Cell Cycle-dependent Control of the universal minicircle sequence (UMS)-binding protein (UMSBP) Redox State Regulates Its Binding to UMS—Previous studies have shown that the steady state levels of UMSBP mRNA and protein are apparently constant throughout the trypanosomatid cell cycle [41], suggesting that regulation of UMSBP activity in vivo is most likely of a post-translational nature
Considering the essential role played by the zinc fingers of UMSBP in the protein interaction with DNA [12], the high sensitivity of zinc fingers to redox changes, and the reversible nature of the changes in the protein’s redox state, we have previously examined the possibility that redox signaling may be involved in the regulation of UMSBP
The question of whether UMSBP activity is regulated in vivo through the control of the protein redox state is being first challenged in the current study
Summary
PCR primers were prepared by Syntezza, Diamide (diazene dicarboxylic acid bis(N,N-dimethylamide)) was purchased from Sigma, trypanothione (N1,N8-bis(glutationyl)spermidine) was from Bachem, and restriction endonucleases and polynucleotide kinase were from MBI Fermentas. Trypanosoma cruzi recombinant trypanothione reductase (TcTRH6) and Crithidia fasciculata recombinant tryparedoxins (CfTXN1H6 and CfTXN2H6) were a generous gift of Dr L. Flohe (Department of Biochemistry, Technical University of Braunschweig, Germany). Pet15b plasmid (Novagene), expressing C. fasciculata tryparedoxin peroxidase was a generous gift of Drs Alan H. Fairlamb and Janine Koening (University of Dundee, Scotland, UK). Anti-Leishmania infantum mitochondrial tryparedoxin peroxidase (LimTXNPx) antibodies were a generous gift of Dr Ana Maria Tomas (Institute for Molecular and Cell Biology, Porto, Portugal), and anti-T. brucei TXNPx (TbTXNPx) antibodies were a generous gift of Dr Marcelo Comini from the Biochemie Zentrum Heidelberg
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